NMR metabolite comparison of local pigmented rice in Yogyakarta


Dio N. Wijaya(1*), Febri Adi Susanto(2), Yekti Asih Purwestri(3), Dyah Ismoyowati(4), Tri Rini Nuringtyas(5)

(1) Faculty of Biology, Universitas Gadjah Mada, Jalan Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia
(2) Research Center for Biotechnology, Universitas Gadjah Mada, Depok, Sleman, Yogyakarta 55281, Indonesia
(3) Faculty of Biology, Universitas Gadjah Mada, Jalan Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia
(4) Agricultural Technology, Universitas Gadjah Mada, Jalan Socia Justicia Bulaksumur, Yogyakarta 55281, Indonesia
(5) Faculty of Biology, Universitas Gadjah Mada, Jalan Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia
(*) Corresponding Author


Pigmented rice may have a black or red color due to higher anthocyanin content in its grain. A natural antioxidant, many studies on anthocyanin have reported its positive effects on human health. This fact has spurred the development of pigmented rice as a functional food. This study aimed to compare the metabolite profiles of black and red rice. Three black rice cultivars, namely Melik, Pari Ireng, and Cempo Ireng Sleman, and two red rice cultivars, Inpari 24 and RC 204, were used. After husk removal, grain samples were ground in liquid nitrogen and dried with a freeze dryer. The dried samples were extracted using 50% MeOD4 (in a D2O phosphate buffer pH 6 containing 0.01% TSP as an internal standard). Metabolomic analysis was performed using 500 MHz NMR followed by multivariate data analysis. An orthogonal partial least squares-discriminant analysis (OPLS-DA) model ađer PCA was constructed to discriminate between the five different cultivars. The resulting OPLS-DA score plot revealed a clear separation between black rice and red rice. The metabolites that could influence the separation of red rice and black rice were valine, threonine, alanine, glutamate, galactinol, β-glucose, α-glucose, raffinose, and fumaric acid.


metabolite comparison; nuclear magnetic resonance; pigmented rice

Full Text:



Andoko A. 2012. Budi Daya Padi Secara Organik [Organic rice cultivation]. Jakarta: Penebar Swadaya.

Asamarai AM, Addis PB, Epley RJ, Krick TP. 1996. Wild Rice Hull Antioxidants. Journal of Agricultural and Food Chemistry 44:126–130. doi:10.1021/jf940651c.

Ashford D, Neuberger A. 1980. 4-Hydroxyl-l-proline in plant glycoproteins: Where does it come from and what is it doing there? Trends in Biochemical Sciences 5:245–248. doi:10.1016/S0968-0004(80)80813-9.

Binder S, Knill T, Schuster J. 2007. Branched-chain amino acid metabolism in higher plants. Physiologia Plantarum 129:68–78. doi:10.1111/j.1399-3054.2006.00800.x.

Bolouri-Moghaddam MR, Le Roy K, Xiang L, Rolland F, Van den Ende W. 2010. Sugar signalling and antioxidant network connections in plant cells: Sugar signalling and antioxidant networks in plants. FEBS Journal 277:2022–2037. doi:10.1111/j.1742-4658.2010.07633.x.

Bylesjö M, Rantalainen M, Cloarec O, Nicholson JK, Holmes E, Trygg J. 2006. OPLS discriminant analysis: combining the strengths of PLS-DA and SIMCA classification. Journal of Chemometrics 20:341–351. doi:10.1002/cem.1006.

Chen Q, Ling W, Ma J, Mei J. 2000. [Effects of black and red rice on the formation of aortic plaques and blood lipids in rabbits]. Wei Sheng Yan Jiu 29:170–172.

Dai H, Xiao C, Liu H, Tang H. 2010. Combined NMR and LC-MS analysis reveals the metabonomic changes in Salvia miltiorrhiza Bunge induced by water depletion. Journal of Proteome Research 9:1460–1475. doi:10.1021/pr900995m.

Fan TW-M. 1996. Metabolite profiling by one- and two-dimensional NMR analysis of complex mixtures. Progress in Nuclear Magnetic Resonance Spectroscopy 28:161–219. doi:10.1016/0079-6565(95)01017-3.

Frank T, Reichardt B, Shu Q, Engel K-H. 2012. Metabolite profiling of colored rice (Oryza sativa L.) grains. Journal of Cereal Science 55:112–119. doi:10.1016/j.jcs.2011.09.009.

Gill SS, Tuteja N. 2010. Polyamines and abiotic stress tolerance in plants. Plant Signaling & Behavior 5:26–33. doi:10.4161/psb.5.1.10291.

Ginanjar E. 2016. Kajian ketahanan padi berpigmen (Oryza sativa L.) terhadap penyakit blas melalui pendekatan transkriptomik dan metabolomik [Transcriptomics and metabolomics study of pigmented rice (Oryza sativa L.) resistant to blast disease] [Master’s thesis]. [Yogyakarta, Indonesia]: Universitas Gadjah Mada.

Hall R, Beale M, Fiehn O, Hardy N, Sumner L, Bino R. 2002. Plant metabolomics: the missing link in functional genomics strategies. Am Soc Plant Biol.

Halliwell B. 1977. Generation of hydrogen peroxide, superoxide and hydroxyl radicals during the oxidation of dihydroxyfumaric acid by peroxidase. The Biochemical Journal 163:441–448.

Heldt H-W, Heldt F. 2004. Plant Biochemistry. 2nd ed. Academic Press.

Heldt H-W, Heldt F. 2005. Plant Biochemistry. 3rd ed. Academic Press.

Herbers K, Meuwly P, Métraux JP, Sonnewald U. 1996. Salicylic acid-independent induction of pathogenesis-related protein transcripts by sugars is dependent on leaf developmental stage. FEBS Letters 397:239–244.

Hofmann J, El Ashry AEN, Anwar S, Erban A, Kopka J, Grundler F. 2010. Metabolic profiling reveals local and systemic responses of host plants to nematode parasitism. The Plant Journal 62:1058–1071. doi:10.1111/j.1365-313X.2010.04217.x.

Huang T, Jander G, de Vos M. 2011. Non-protein amino acids in plant defense against insect herbivores: Representative cases and opportunities for further functional analysis. Phytochemistry 72:1531–1537. doi:10.1016/j.phytochem.2011.03.019.

Huang Y-P, Lai H-M. 2016. Bioactive compounds and antioxidative activity of colored rice bran. Journal of Food and Drug Analysis 24:564–574. doi:10.1016/j.jfda.2016.01.004.

Hyun JW, Chung HS. 2004. Cyanidin and Malvidin from Oryza sativa cv. Heugjinjubyeo mediate cytotoxicity against human monocytic leukemia cells by arrest of G(2)/M phase and induction of apoptosis. . Journal of Agricultural and Food Chemistry 52:2213–2217. doi:10.1021/jf030370h.

Ichikawa H, Ichiyanagi T, Xu B, Yoshii Y, Nakajima M, Konishi T. 2001. Antioxidant Activity of Anthocyanin Extract from Purple Black Rice. Journal of Medicinal Food 4:211–218. doi:10.1089/10966200152744481.

Jones OAH, Maguire ML, Griffin JL, Jung Y-H, Shibato J, Rakwal R, Agrawal GK, Jwa N-S. 2011. Using metabolic profiling to assess plant-pathogen interactions: an example using rice (Oryza sativa) and the blast pathogen Magnaporthe grisea. European Journal of Plant Pathology 129:539–554. doi:10.1007/s10658-010-9718-6.

Kim HK, Choi YH, Verpoorte R. 2010. NMR-based metabolomic analysis of plants. Nature Protocols 5:536–549. doi:10.1038/nprot.2009.237.

Kim HY, Hwang IG, Kim TM, Woo KS, Park DS, Kim JH, Kim DJ, Lee J, Lee YR, Jeong HS. 2012. Chemical and functional components in different parts of rough rice (Oryza sativa L.) before and after germination. Food Chemistry 134:288–293. doi:10.1016/j.foodchem.2012.02.138.

Kim MS, Cho SM, Kang EY, Im YJ, Hwangbo H, Kim YC, Ryu C-M, Yang KY, Chung GC, Cho BH. 2008. Galactinol is a signaling component of the induced systemic resistance caused by Pseudomonas chlororaphis O6 root colonization. Molecular Plant-Microbe Interactions 21:1643–1653.

Koch K. 2004. Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Current Opinion in Plant Biology 7:235–246. doi:10.1016/j.pbi.2004.03.014.

Komatsuzaki N, Tsukahara K, Toyoshima H, Suzuki T, Shimizu N, Kimura T. 2007. Effect of soaking and gaseous treatment on GABA content in germinated brown rice. Journal of Food Engineering 78:556–560. doi:10.1016/j.jfoodeng.2005.10.036.

Kristamtini, Purwaningsih H. 2009. Potensi pengembangan beras merah sebagai plasma nutfah Yogyakarta [Development potential of red rice as Yogyakarta’s germplas]. Jurnal Litbang Pertanian 28:88--95.

Kusano M, Yang Z, Okazaki Y, Nakabayashi R, Fukushima A, Saito K. 2015. Using metabolomic approaches to explore chemical diversity in rice. Molecular Plant 8:58–67. doi:10.1016/j.molp.2014.11.010.

Ling WH, Cheng QX, Ma J, Wang T. 2001. Red and black rice decrease atherosclerotic plaque formation and increase antioxidant status in rabbits. J. Nutr. 131:1421–1426.

Ling WH, Wang LL, Ma J. 2002. Supplementation of the black rice outer layer fraction to rabbits decreases atherosclerotic plaque formation and increases antioxidant status. Journal of Nutrition 132:20–26.

Liu C, Du B, Hao F, Lei H, Wan Q, He G, Wang Y, Tang H. 2017. Dynamic metabolic responses of brown planthoppers towards susceptible and resistant rice plants. Plant Biotechnology Journal 15:1346–1357. doi:10.1111/pbi.12721.

Matsumoto H, Nakamura Y, Tachibanaki S, Kawamura S, Hirayama M. 2003. Stimulatory effect of cyanidin 3-glycosides on the regeneration of rhodopsin. Journal of Agricultural and Food Chemistry 51:3560–3563. doi:10.1021/jf034132y.

Mills GC, Alperin JB, Trimmer KB. 1975. Studies on variant glucose-6-phosphate dehydrogenases: G6PD Fort Worth. Biochemical Medicine 13:264–275.

Mingwei Z, Baojiang G, Ruifen Z, Jianwei C, Zhencheng W, Zhihong X, Yan Z, Xiaojun T. 2006. Separation, purification and identification of antioxidant compositions in black rice. Agricultural Sciences in China 6:431--440.

Moreno YS, Sánchez GS, Hernández DR, Lobato NR. 2005. Characterization of anthocyanin extracts from maize kernels. Journal of Chromatographic Science 43:483–487.

Morkunas I, Marczak Ł, Stachowiak J, Stobiecki M. 2005. Sucrose-induced lupine defense against Fusarium oxysporum. Sucrose-stimulated accumulation of isoflavonoids as a defense response of lupine to Fusarium oxysporum. Plant Physiology and Biochemistry 43:363–373. doi:10.1016/j.plaphy.2005.02.011.

Na Jom K, Lorjaroenphon Y, Udompijitkul P. 2016. Differentiation of Four Varieties of Germinating Thai Colored Indica Rice (Oryza sativa L.) by Metabolite Profiling. Food Science and Technology Research 22:65–73. doi:10.3136/fstr.22.65.

Nam MH, Bang E, Kwon TY, Kim Y, Kim EH, Cho K, Park WJ, Kim B-G, Yoon IS. 2015. Metabolite profiling of diverse rice germplasm and identification of conserved metabolic markers of rice roots in response to long-term mild salinity stress. International Journal of Molecular Sciences 16:21959–21974. doi:10.3390/ijms160921959.

Nam SH, Choi SP, Kang MY, Koh HJ, Kozukue N, Friedman M. 2006. Antioxidative activities of bran extracts from twenty one pigmented rice cultivars. Food Chemistry 94:613–620. doi:10.1016/j.foodchem.2004.12.010.

Nuringtyas TR, Choi YH, Verpoorte R, Klinkhamer PGL, Leiss KA. 2012. Differential tissue distribution of metabolites in Jacobaea vulgaris, Jacobaea aquatica and their crosses. Phytochemistry 78:89–97. doi:10.1016/j.phytochem.2012.03.011.

Pramai P, Abdul Hamid NA, Mediani A, Maulidiani M, Abas F, Jiamyangyuen S. 2017. Metabolite profiling, antioxidant, and α-glucosidase inhibitory activities of germinated rice: nuclear-magnetic-resonance-based metabolomics study. Journal of Food and Drug Analysis. doi:10.1016/j.jfda.2016.11.023.

Rolland F, Baena-Gonzalez E, Sheen J. 2006. Sugar sensing and signaling in plants: conserved and novel mechanisms. Annual Review of Plant Biology 57:675–709. doi:10.1146/annurev.arplant.57.032905.105441.

Sakamoto A, Murata N. 2002. The role of glycine betaine in the protection of plants from stress: clues from transgenic plants. Plant, Cell & Environment 25:163–171.

Salinas Moreno Y, Sánchez GS, Hernández DR, Lobato NR. 2005. Characterization of anthocyanin extracts from maize kernels. Journal of Chromatographic Science 43:483–487.

Satyanarayana T, Radhakrishnan AN. 1962. Biosynthesis of valine and isoleucine in plants. Biochimica et Biophysica Acta 56:197–199. doi:10.1016/0006-3002(62)90554-1.

Slamet-Loedin I, Rahayu W, Hutajulu S, Wibowo J. 1997. Penggunaan dua strain Agrobacterium tumefaciens supervirulen untuk ko-kultivasi tanaman padi kultivar Cisadane dan Rojolele [The use of two strains Agrobacterium tumafaciens supervirulen for co-cultivation of rice cultivars Cisadane and Rojolele]. Surabaya.

Steinbrenner AD, Gómez S, Osorio S, Fernie AR, Orians CM. 2011. Herbivore-induced changes in tomato (Solanum lycopersicum) primary metabolism: a whole plant perspective. Journal of Chemical Ecology 37:1294–1303. doi:10.1007/s10886-011-0042-1.

Stuttmann J, Hubberten H-M, Rietz S, Kaur J, Muskett P, Guerois R, Bednarek P, Hoefgen R, Parker JE. 2011. Perturbation of Arabidopsis amino acid metabolism causes incompatibility with the adapted biotrophic pathogen Hyaloperonospora arabidopsidis. Plant Cell 23:2788–2803. doi:10.1105/tpc.111.087684.

Tananuwong K, Tewaruth W. 2010. Extraction and application of antioxidants from black glutinous rice. LWT - Food Science and Technology 43:476–481. doi:10.1016/j.lwt.2009.09.014.

Uawisetwathana U, Graham SF, Kamolsukyunyong W, Sukhaket W, Klanchui A, Toojinda T, Vanavichit A, Karoonuthaisiri N, Elliott CT. 2015. Quantitative 1H NMR metabolome profiling of Thai Jasmine rice (Oryza sativa) reveals primary metabolic response during brown planthopper infestation. Metabolomics 11:1640–1655. doi:10.1007/s11306-015-0817-4.

Wang Q, Han P-H, Zhang M-W, Xia M, Zhu H-L, Ma J, Hou M-J, Tang Z-H, Ling W-H. 2007. Supplementation of black rice pigment fraction improves antioxidant and anti-inflammatory status in patients with coronary heart disease. Asia Pacific Journal of Clinical Nutrition 16:295–301.

Westerhuis JA, Velzen EJJ van, Hoefsloot HCJ, Smilde AK. 2010. Multivariate paired data analysis: multilevel PLSDA versus OPLSDA. Metabolomics 6:119–128. doi:10.1007/s11306-009-0185-z.

Wijaya D. 2017. Kajian ketahanan padi berpigmen (Oryza sativa L.) terhadap walang sangit (Leptocorisa oratorius) berbasis NMR metabolomik [NMR metabolomics study on rice ear bug (Leptocorisa oratorius) resistance in pigmented rice] [Master’s thesis]. [Yogyakarta, Indonesia]: Universitas Gadjah Mada.

Xu F, Wang E. 1989. The effects of pigmented rice on hemoglobin regeneration in anemic rats. Acta Nutr Sin. 11:120--125.

Yawadio R, Tanimori S, Morita N. 2007. Identification of phenolic compounds isolated from pigmented rices and their aldose reductase inhibitory activities. Food Chemistry 101:1616–1625. doi:10.1016/j.foodchem.2006.04.016.

Yoshinaga K, Takahashi K, Yoshizawa K. 1986. Liqueur making using pigments of red rice. Journal of the Society of Brewing, Japan 81:337–340. doi:10.6013/jbrewsocjapan1915.81.337.

DOI: https://doi.org/10.22146/ijbiotech.27308

Article Metrics

Abstract views : 3909 | views : 3323


  • There are currently no refbacks.

Copyright (c) 2017 The Author(s)

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.